Structural characterization of recombinant hepatitis E virus ORF2 proteins in baculovirus-infected insect cells.
The hepatitis E virus (HEV) capsid antigen has been proposed as a candidate subunit vaccine for the prevention of hepatitis E. The full-length HEV ORF2 protein product is predicted to contain 660 amino acids and to weigh 72,000 daltons. Expression of the HEV ORF2 capsid gene from recombinant baculoviruses in insect cells produced multiple immunoreactive proteins ranging in size from 30 to 100 kDa. The most abundant HEV proteins had molecular weights of 72, 63, 56, and 53 kDa. Temporal expression kinetics of these viral polypeptides indicated that the 72- and 63-kDa polypeptides were produced abundantly within the initial 36 h. postinfection but were replaced by 56- and 53-kDa polypeptides in the cell and medium, respectively, by 48 h postinfection. The 53-kDa protein was secreted as early as 24 h. postinfection, and accumulation in the medium peaked by 72 h postinfection. Purification of the 53-, 56-, and 63-kDa viral polypeptides was accomplished by anion-exchange and subsequent gel filtration chromatography. Sequence analysis of the 53-, 56-, and 63-kDa HEV polypeptides indicated that the amino terminus was amino acid residue 112 of the predicted full-length protein product. The results of carboxy terminal amino acid sequencing indicated that the carboxy terminus of the 53-, 56-, and 63-kDa HEV proteins was located at amino acid residues 578, 607, and 660, respectively. The molecular masses of the 53- and 56-kDa HEV polypeptides were 53,872 and 56,144 as determined by mass spectroscopy.
Robinson
R A
RA
Molecular Virology Laboratory, DynCorp, 1 Taft Court, Rockville, Maryland 20850, USA. robinsor@erols.com
Burgess
W H
WH
Emerson
S U
SU
Leibowitz
R S
RS
Sosnovtseva
S A
SA
Tsarev
S
S
Purcell
R H
RH
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000956:Antigens, Viral / Q000737:chemistry / Q000235:genetics; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002213:Capsid / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D002999:Clone Cells; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D016751:Hepatitis E / Q000517:prevention & control; D016752:Hepatitis E virus / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D018411:Spodoptera; D014761:Viral Hepatitis Vaccines / Q000235:genetics / Q000302:isolation & purification; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
D000596:Amino Acids; D000956:Antigens, Viral; D017931:DNA Primers; D011994:Recombinant Proteins; D014761:Viral Hepatitis Vaccines; D014764:Viral Proteins